Ionizing radiation (IR) and inter-strand cross-linking agents (ICL) induce DNA double-stranded breaks (DSB). DSB are the most harmful type of DNA damage, which cause genome instability, cancer, genetic diseases, and premature aging. The system of homologous recombination (HR) is responsible for the repair of DSB repair in humans. However, DSB-inducing agents, IR and ICL, are also used in anti-cancer therapy. In order to increase the efficiency of this therapy, we propose to suppress HR in cancer cells by using specific inhibitors against a key human HR protein, Rad51 (hRad51). These inhibitors will help to develop novel combination therapies that allow to reduce doses of IR and ICL thereby decreasing their cytotoxicity. Specific inhibitors and stimulators of hRad51 will also present a valuable tool in fundamental studies on the mechanisms of HR in human cells. Therefore, the Aim of this proposal is to identify specific inhibitors and stimulators of hRad51 using high throughput screening (HTS) of the molecular libraries of reagents. hRad51 possesses a unique activity, it promotes DNA strand exchange between homologous DNA molecules, a basic step of HR. We have developed a fluorimetric assay that allows to measure DNA strand exchange activity of hRad51 in vitro. We will use this assay for the primary HTS. The selected compounds will be validated using conventional radioactively-based DNA strand exchange assays (secondary screen).Using microbial hRad51 homologues and structurally unrelated hRad54 protein we will determine the selectivity and specificity of the selected compounds. In continuation of this grant, we will employ the selected compounds for analysis of the mechanisms of HR in humans. The therapeutic potential the prioritized compounds will be examined using reconstituted HR system in vitro, transformed human cells, and immuno-deficient mice with transplanted human xenografts (SCID-hu mice). [unreadable] [unreadable] [unreadable]